1Sidney-Kimmel Comprehensive Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, United States.

Abstract

The mammalian target of rapamycin (mTOR) is an evolutionarily conserved kinase which plays a role in integrating environmental cues. mTOR signals via two complexes: TORC1, which contains the Regulatory Associated Protein of TOR (raptor), and TORC2, which contains the Rapamycin-insensitive Companion of TOR (rictor). The immunosuppressive/anti-cancer agent rapamycin inhibits TORC1 function by disrupting the mTOR-raptor interaction. In an effort to understand the downstream consequences of TORC1 activation in T cells we performed a proteomic analysis of raptor binding proteins. Using this approach we have identified Hsp90 as an activation-induced binding partner of raptor in T cells. Pharmacologic inhibition of Hsp90 leads to a decrease in raptor expression and TORC1 activity. Furthermore, full T cell activation during Hsp90 blockade leads to T cell tolerance in the form of anergy. Overall, our findings suggest that Hsp90 inhibitors might represent a novel means of promoting T cell tolerance.

(A) Rapamycin induces anergy in the presence of costimulation. A.E7 Th1 cells were stimulated overnight with α-CD3, α-CD3 and α-CD28, or α-CD3, α-CD28, and rapamycin. Cells were then washed and rested in media for five days and restimulated with α-CD3 and α-CD28. Error bars indicate S.D. Results are representative of five independent experiments. (B) Rapamycin inhibits TORC1 assembly. Cells were mock or α-CD3 + α-CD28 stimulated in the presence of rapamycin or 17-AAG, lysates were made and immunoprecipitation (IP) for mTOR was performed. Immunoblots (IB) were performed on the IPs for the presence of raptor. IBs are representative of three independent experiments. (C) Proteomic strategy for identifying TORC1 substrates. Jurkat T cells were either incubated in serum-free media (to reduce the amount of basal mTOR activation from serum) or stimulated with 500 uM pervanadate and 20 nM caliculin A. The cells were then lysed and subjected to an IP of raptor. The two lysates were separated by SDS-PAGE and silver stained. We then looked for protein bands that were differentially bound to raptor in the lysate from stimulated versus unstimulated cells. One band identified near 90 kDa was excised, digested with trypsin, extracted and analyzed by nanospray LCMS/MS (top panel). The raw LCMS/MS data was analyzed using the MASCOT search engine against the NCBInr human database; two peptide sequences were identified and matched to Hsp90 (bottom panel, sequence in red). (D) Hsp90 and raptor interact in a TCR-induced manner. 5C.C7 primary Th1 cells were serum-starved for 3 hours, then left unstimulated or given α-CD3 and α-CD28 for 3 hours, lysed and subjected to the IP indicated. IPs were then probed for the binding partner. IBs are representative of three independent experiments.

Hsp90 inhibition in T cells leads to the development of an anergic state

(A) Raptor is a client of Hsp90, which is necessary for TORC1 signaling. A.E7 Th1 cells were stimulated in the presence of rapamycin (200nM), 17-AAG (200nM), radicicol (10μM), or CCT018159 (5μM), then probed for raptor and phospho-S6K1 by IB. rictor (the TORC2 adaptor) is included as a loading control. IBs are representative of three independent experiments. (B) A.E7 Th1 cells were stimulated with α-CD3, α-CD3 and α-CD28, or α-CD3, α-CD28, and drug overnight, after which IL-2 production was measured by ELISA from a sample of supernatant (induction). (C) Cells from B were then washed and rested in unsupplemented fresh media (no drug). After 5 days, some cells were removed and probed for raptor and rictor by IB, to confirm raptor levels have returned. (D) The remainder of the cells were stimulated with α-CD3 and α-CD28 (rechallenge with no drug) and interrogated for IL-2 production by ELISA. Results are pooled from three independent experiments. (E) Supernatants from D were interrogated for IFN-γ production by ELISA. Results are pooled from three independent experiments. (F) Cells restimulated in D were pulsed with 3H-thymidine and assayed for proliferation. Error bars indicate S.D. Results are representative of three independent experiments.